The Characteristic Of Apoptosis Of T Lymphocytes In Peritoneal Fluid And Peripheral Blood And The Effect Of Soluble Fas On T Lymphocytes Apoptosis In The Patients With Epithelial Ovarian Cancer

It is well known that patients with advanced ovarian carcinoma have progressively impaired immune responses and have a high mortality.Treatment and prognosis are not optimistic for ovarian carcinoma which emerges secretly and examine late, although the chemistry theropy and operation is progressing.Presently,inhibiting immune escape and counterattack has become a hot topic in the field of cancer immunologic treatment.Effective immunologic treatment is based on full comprehension of the state of impaired immune responses and correlated factors.Now,there are several studies about the apoptosis and function of tumor-infiltration lymphocyte in patients with cancer including ovarian cancer.Extensive dissemination of ovarian cancer in the peritoneal cavity is more common than distant metastasis,and it was shown that the phaenotype of peritoneal fluid T lymphocytes of ovarian cancer patients differed from that of gastroenteric cancer patients.Primary ovarian cancer may have special mechanism of immunologic injury in abdominal cavity,and it will be helpful to understand the characteristic of apoptosis of T lymphocytes in peritoneal fluid and peripheral blood and the relationship between T lymphocytes apoptosis and correlated factors in the patients with epithelial ovarian cancer. Fas/APO-1/CD95 is a type of membrane proteins and a member of the tumor necrosis factor-R4 superfamilies.The interaction between FasL and cell-surface Fas induces cellular apoptosis.Fas exists both as a cell-surface protein(mFas)and as a soluble protein(sFas)which lacks a transmembrane domain and is generated either by proteolytic cleavage of membrane-bound fas or by alternative mRNA splicing,sFas can inhibit Fas-FasL binding and block Fas-mediated apoptosis of carcinoma cells,so it may play a role in tumorogenesis.Being different from patients with many other solid tumors in abdominal cavity,ovarian cancer has the special feature of extensive dissemination in abdominal cavity rather than distant metastasis,but there are few studies about the level of sFas in peritoneal fluids and the influence of sFas on this biological behavior of ovarian carcinoma.Malignant diseases are known to be associated with immune suppression,which facilitates the escape of tumors from the host immune system.Carcinoma cells co-opt several physiologic regulatory mechanisms to resist immune attack.It has been suggested that one such carcinoma-corrupted immunologic checkpoint may be tumor cell expression of Fas ligand(FasL)to induce Fas-sensitive infiltrating T(and natural killer)effector cells to apoptosis.Therefore,the next logical question is wether sFas can block Fas-mediated apoptosis of T cell in patients with carcinoma.However,the effect of sFas on the apoptosis of lymphocytes is still controversial.The present study aims at investigating the feature of immune suppression and the role of sFas in the apoptosis of T cell in patients with ovarian carcinoma.ELISA, immunoprecipitation,Cells culture and Flow cytometry technique are used in this study.The study was divided into two parts:1.Study of apoptosis of peritoneal fluid and peripheral blood T lymphocytes in patients with epithelial ovarian cancer and the relationship with correlated factors,such as Fas expression,CA125 and sFas level.2. Study of the effect of sFas on Jurkat cell apoptosis. Part oneApoptosis of peritoneal fluid and peripheral blood T lymphocytes in patients with epithelial ovarian cancer and the relationship with correlated factors1.The characteristic of apoptosis of T lymphocytes in peritoneal fluid and peripheral blood and the relationship between T lymphocytes apoptosis and Fas expression,CA125 in the patients with epithelial ovarian cancerMethods:Apoptosis and Fas expression of peritoneal fluid and peripheral blood T lymphocytes were assessed by flow cytometry.Samples were obtained from the following objects respectively:patients with stageⅢ-Ⅳovarian cancer(n=18) before and after treatment,patients with stageⅠ-Ⅱovarian cancer(n=16),patients with benign ovarian tumor(n=19),patients with Krukenberg tumor(n=6)and normal control(n=21).Peritoneal fluids were obtained from all the patients with ovarian cancer,Krukenberg tumor and ten patients with benign ovarian tumor.Level of serum CA125 was assessed for patients with ovarian cancer.Results:In the patients with stageⅢ～Ⅳovarian cancer,apoptosis level of peripheral blood T lymphocytes was 5.81(4.08～10.18)%,significantly higher than those from patients with stageⅠ～Ⅱovarian cancer,patients with benign ovarian tumor, control(P＜0.008 in all instances)and patients with stageⅢ～Ⅳovarian cancer after treatment(P＜0.05).The positive rate and intensity of Fas expression on peripheral blood T lymphocytes from patients with stageⅢ～Ⅳovarian cancer were (44.36±6.07)%and 49.51±5.72,significantly higher than those from control(P＜0.05). In peritoneal fluid,the apoptosis of T lymphocytes,the positive rate and intensity of Fas expression on T lymphocytes from patients with stageⅠ～Ⅱand stageⅢ～Ⅳovarian cancer were 17.41(7.97～30.95)%,(54.13±13.39)%,55.16±9.12 and 34.06(16.33±43.64)%,(62.23±11.69)%,67.70±23.65,respectively,increased significantly compared with those from patients with benign ovarian tumor,which were 0.89(0.71～1.76)%,(35.92±6.05)%,41.88±6.07,respectively(P＜0.01 in all instances).The apoptosis level and the positive rate of Fas expression on peritoneal fluid T lymphocytes from patients with stageⅢ～Ⅳovarian cancer were significantly higher than those from patients with Krukenberg tumor(P＜0.05 in all instances).The apoptosis levels of T lymphocytes had a positive correlation with the positive rate and intensity of Fas expression on T cells in the peritoneal fluid from ovarian carcinoma. (r=0.534,P=0.000;r=0.594,P=0.000).There was a positive correlation between the serum CA125 level and the apoptosis level of peritoneal fluid T cell in patients with stageⅠ～Ⅱovarian cancer(r=0.801,P=0.000).For ovarian cancer,the apoptosis level of peritoneal fluid T lymphocytes from patients with serum CA125＞500KU/Lwas higher than that from patients with serum CA125≤500KU/L(P＜0.05).2.The correlation between soluble Fas level and apoptosis of T cells in ovarian carcinomaMethods:Apoptosis of T cell from peripheral blood and peritoneal fluids were assessed by flow cytometry.Soluble Fas level was assayed by ELISA Kit.Samples were obtained from the following objects respectively:patients with stageⅢ～Ⅳovarian cancer(n=22),patients with stageⅠ～Ⅱovarian cancer(n=10),patients with benign ovarian tumor(n=8).Results:The sFas level and apoptosis of T cells in peripheral blood and peritoneal fluid from stageⅢ-Ⅳovarian carcinoma were significantly higher than those from stageⅠ-Ⅱovarian carcinoma(P＜0.01 in all instances)and benign ovarian tumor (P＜0.01 in all instances).In peritoneal fluid,sFas level and apoptosis of T cells from stageⅠ-Ⅱovarian carcinoma were significantly higher than those from benign ovarian tumor(P＜0.01 in all instances).There was a positive correlation between the sFas level and the apoptosis of T cells in peritoneal fluids from stageⅢ-Ⅳovarian carcinoma(r=0.647,P=0.001). Part twoThe effect of Recombinant Human sFas and sFas in peritoneal fluid from ovarian cancer on Jurkat cell apoptosisMethods:ELISA,cell culture,immunoprecipitation,MTT and flow cytometry were used to study the effect of sFas on Jurkat cell apoptosis.Results:Recombinant Human sFas can not affect the growth and apoptosis of Jurkat cell significantly.Peritoneal fluid of ovarian carcinoma could induce Jurkat cell apoptosis significantly.The blocking of Fas expression on Jurkat cell surface,but not deletion of sFas,could remarkably restrain the apoptosis level.Conclusion:1.In ovarian carcinoma patients,apoptosis of T cells increased significantly.The characteristic of apoptosis of T lymphocytes was as follows:Firstly,apoptosis of T cells in peritoneal fluid increased earlier than that in peripheral blood.Secondly,in contrast with Krukenberg tumor,patients with advanced ovarian cancer had higher percentage of apoptotic peritoneal fluid T lymphocytes.It was suggested that the characteristic of apoptosis of T lymphocytes was one of the reasons why extensive dissemination of overain cancer in the peritoneal cavity is more common than distant metastasis.2.In ovarian carcinoma patients,the Fas expression on T cells increased significantly. The Fas expression on T lymphocytes had a positive correlation with the apoptosis of T cells.It was shown that ovarian cancer may induce T cells apoptosis by means of upregulate the Fas expression on T cells.3.Level of serum CA125 can reflect the celiac immunity defection in patients with ovarian cancer.4.The sFas level and the apoptosis of T cells in peripheral blood and peritoneal fluid from ovarian carcinoma increased synchronously.The sFas level had a positive correlation with the apoptosis of T cells in the peritoneal fluid from advanced ovarian carcinoma.It was suggested that elevated sFas is correlated with apoptosis of T cells.5.The peritoneal fluid of ovarian carcinoma patients could induce Jurkat cells apoptosis,but the deletion of sFas did not affect apoptosis level of Jurkat cells significantly.Furthermore,Recombinant Human sFas can not induce Jurkat cell apoptosis in vitro.In contrast,the blocking of cell-surface Fas on Jurkat cell can remarkably restrain Jurkat cell apoptosis.It was suggested that Fas pathway play an important role in the apoptosis induction and sFas be not found to induce or inhibit T cell apoptosis.6.Level of sFas can reflect the apoptotic state of peritoneal fluid T lymphocytes in patients with ovarian cancer.